Search

The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while β/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (Na V ) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that β/δ-Uf2a is a potent modulator of human Na V 1.5 (EC 50 : 55 nM), Na V 1.6 (EC 50 : 0.86 nM), and Na V 1.7 (EC 50 : 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The use of enzymes for the site-specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP-catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving-group peptide. Our metal-complexation-based strategy enables efficient peptide/protein labeling at the N- or C-terminus with near-equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein-protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable., (© 2020 Wiley-VCH GmbH.)

Voltage-gated sodium (Na V ) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal Na V channels inhibiting peak current of hNa V 1.1, rNa V 1.2, hNa V 1.6, and hNa V 1.7 while concurrently inhibiting fast inactivation of hNa V 1.1 and rNa V 1.3. The DII and DIV S3-S4 loops of Na V channel voltage sensors are important for the interaction of Pre1a with Na V channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between Na V channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a's potent and selective inhibitory effect on the fast inactivation process of Na V 1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of Na V channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.

Loss-of-function mutations of Na(V)1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of Na(V)1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na(V)1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na(V)1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na(V)1.7 inhibitors and significantly reduced in Na(V)1.7(-/-) mice. To validate the use of the model for profiling Na(V)1.7 inhibitors, we determined the Na(V) selectivity and tested the efficacy of the reported Na(V)1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited Na(V)1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na(V)1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited Na(V) channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na(V)1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na(V)1.7 inhibitors.

Peptides are attracting increasing interest from the pharmaceutical industry because of their specificity and ability to address novel targets, including protein-protein interactions. However, typically they require stabilization for therapeutic applications owing to their susceptibility to degradation by proteases. Advances in the ability to chemically synthesize peptides and the development of new side-chain and backbone ligation strategies provide new tools to stabilize bioactive peptide epitopes. Two such epitopes are LyP1, a nine residue peptide that localizes to tumor cells and has potential as an anticancer therapeutic, and RGDS, a tetrapeptide shown to bind to survivin and induce apoptosis. Here we applied a variety of strategies for the stabilization of LyP1 and RGDS, including side-chain cyclization using "click" chemistry and "grafting" the epitopes into two naturally occurring cyclic peptide scaffolds, i.e., θ-defensins and cyclotides. NMR data showed that the three-disulfide θ-defensin and cyclotide scaffolds accommodated the LyP1 and RGDS epitopes but that scaffolds with fewer disulfide bonds were structurally compromised by inclusion of the LyP1 epitope. LyP1, LyP1-, and RGDS-grafted peptides that were largely unstructured also had reduced resistance to degradation in human serum, showing that grafting into a stable cyclic scaffold is an effective strategy for increasing the stability of a bioactive peptide epitope. Overall, the study demonstrates several methods for stabilizing peptide epitopes using side-chain or backbone cyclization and illustrates their potential in peptide drug design., (© 2015 Wiley Periodicals, Inc.)

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The synthesis of proteins by native chemical ligation greatly enhances the application of chemistry to complex molecules such as proteins. The essential building blocks for this approach traditionally have been peptide-thioester segments that are linked chemoselectively in consecutive reactions. By using peptide selenoesters instead of thioesters, the ligation rate can be significantly accelerated permitting couplings at difficult sites and potentially enabling new ligation strategies. To facilitate the routine synthesis of selenoester peptides, a general and straightforward procedure has been developed that generates a suitably functionalized resin from which the desired selenoester peptide can be readily synthesized. This simple approach utilizes readily available and cheap chemical agents and enables production of peptide selenoesters of excellent quality in short time and with high recovery. In addition, the stability of peptide selenoesters was examined under different native chemical ligation conditions and compared to thioesters. Selenoesters are slightly more reactive and more susceptible to hydrolysis and aminolysis than thioesters but sufficiently stable under mildly acidic conditions (pH 6.5). Under these conditions, rapid selenoester-mediated ligation is kinetically favoured., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Ziconotide (ω‐conotoxin MVIIA) is an approved analgesic for the treatment of chronic pain. However, the need for intrathecal administration and adverse effects have limited its widespread application. Backbone cyclization is one way to improve the pharmaceutical properties of conopeptides, but so far chemical synthesis alone has been unable to produce correctly folded and backbone cyclic analogues of MVIIA. In this study, an asparaginyl endopeptidase (AEP)‐mediated cyclization was used to generate backbone cyclic analogues of MVIIA for the first time. Cyclization using six‐ to nine‐residue linkers did not perturb the overall structure of MVIIA, and the cyclic analogues of MVIIA showed inhibition of voltage‐gated calcium channels (CaV2.2) and substantially improved stability in human serum and stimulated intestinal fluid. Our study reveals that AEP transpeptidases are capable of cyclizing structurally complex peptides that chemical synthesis cannot achieve and paves the way for further improving the therapeutic value of conotoxins. [ABSTRACT FROM AUTHOR]

Bioactive peptides are a highly abundant and diverse group of molecules that exhibit a wide range of structural and functional variation. Despite their immense therapeutic potential, bioactive peptides have been traditionally perceived as poor drug candidates, largely due to intrinsic shortcomings that reflect their endogenous heritage, i.e., short biological half-lives and poor cell permeability. In this review, we examine the utility of molecular engineering to insert bioactive sequences into constrained scaffolds with desired pharmaceutical properties. Applying lessons learnt from nature, we focus on molecular grafting of cyclic disulfide-rich scaffolds (naturally derived or engineered), shown to be intrinsically stable and amenable to sequence modifications, and their utility as privileged frameworks in drug design. [ABSTRACT FROM AUTHOR]

Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function. [ABSTRACT FROM AUTHOR]

Transpeptidase‐catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N‐to‐C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l‐/d‐ or α‐/β‐amino acid junctions, we find C‐terminal Leu‐ethylenediamine (Leu‐Eda) motifs to be bona fide mimetics of native N‐terminal Gly‐Leu sequences. Appending a C‐terminal Leu‐Eda to synthetic peptides or, via an intein‐splicing approach, to recombinant proteins enables direct transpeptidase‐catalyzed C‐to‐C ligations. This work significantly expands the synthetic scope of enzyme‐catalyzed protein transpeptidation reactions. [ABSTRACT FROM AUTHOR]

Ruthenium‐catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5‐disubstituted 1,2,3‐triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross‐linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor‐1. NMR and X‐ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole‐bridged peptides also displayed superior half‐lives in liver S9 stability assays compared to disulfide‐bridged peptides. This work establishes a foundation for the application of 1,5‐disubstituted 1,2,3‐triazoles as disulfide mimetics. [ABSTRACT FROM AUTHOR]

Cyclotides are plant-derived peptides characterized by a head-to-tail cyclic backbone and a cystine knot motif comprised of three disulfide bonds. Formation of this motif via in vitro oxidative folding can be challenging and can result in misfolded isomers with nonnative disulfide connectivities. Here, we investigated the effect of β-turn nucleation on cyclotide oxidative folding. Two types of β-turn mimics were grafted into kalata B1, individually replacing each of the four β- turns in the folded cyclotide. Insertion of D-Pro-Gly into loop 5 was beneficial to the folding of both cyclic kB1 and a linear form of the peptide. The linear grafted analog folded fourtimes faster in aqueous conditions than cyclic kB1 in optimized conditions. Additionally, the cyclic analogue folded without the need for redox agents by transitioning through a native-like intermediate that was on-pathway to product formation. Kalata B1 is from the Möbius subfamily of cyclotides. Grafting D-Pro-Gly into loop 5 of cyclotides from two other subfamilies also had a beneficial effect on folding. Our findings demonstrate the importance of a β-turn nucleation site for cyclotide oxidative folding, which could be adopted as a chemical strategy to improve the in vitro folding of diverse cystine-rich peptides. [ABSTRACT FROM AUTHOR]

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide. [ABSTRACT FROM AUTHOR]

Innentitelbild: Application and Structural Analysis of Triazole-Bridged Disulfide Mimetics in Cyclic Peptides (Angew. Keywords: disulfide mimetics; inhibitors; peptides; peptidomimetics; triazole bridges EN disulfide mimetics inhibitors peptides peptidomimetics triazole bridges 11258 11258 1 07/03/20 20200706 NES 200706 B 1,5-Disubstituierte 1,2,3-Triazole b können als Mimetika von Disulfidbindungen genutzt werden. Disulfide mimetics, inhibitors, peptides, peptidomimetics, triazole bridges. [Extracted from the article]

μ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of μ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of μ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical μ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (NaV), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of NaV1.4 channels, but the same NaV selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications. [ABSTRACT FROM AUTHOR]

The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while β/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that β/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides. [ABSTRACT FROM AUTHOR]

Disulfide-rich cyclic peptides have exciting potential as leads or frameworks in drug discovery; however, their use is faced with some synthetic challenges, mainly associated with construction of the circular backbone and formation of the correct disulfides. Here we describe a simple and efficient Fmoc solid-phase peptide synthesis (SPPS)-based method for synthesizing disulfide-rich cyclic peptides. This approach involves SPPS on 2-chlorotrityl resin, cyclization of the partially protected peptide in solution, cleavage of the side-chain protecting groups, and oxidization of cysteines to yield the desired product. We illustrate this method with the synthesis of peptides from three different classes of cyclic cystine knot motif-containing cyclotides: Möbius (M), trypsin inhibitor (T), and bracelet (B). We show that the method is broadly applicable to peptide engineering, illustrated by the synthesis of two mutants and three grafted analogues of kalata B1. The method reduces the use of highly caustic and toxic reagents and is better suited for high-throughput synthesis than previously reported methods for producing disulfide-rich cyclic peptides, thus offering great potential to facilitate pharmaceutical optimization of these scaffolds. [ABSTRACT FROM AUTHOR]

The article focuses on stinging trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. It mentions that dendrocnide-induced acute pain lasts for several hours, and intermittent painful flares can persist for days and weeks. It also mentions that pharmacological activity has been attributed to small-molecule neurotransmitters and inflammatory mediators.

Small macrocyclic peptides are promising candidates for new anti-infective drugs. To date, such peptides have been poorly studied in the context of anti-virulence targets. Using phage display and a self-designed peptide library, we identified a cyclic heptapeptide that can bind the carbon storage regulator A (CsrA) from Yersinia pseudotuberculosis and displace bound RNA. This disulfide-bridged peptide, showed an IC50 value in the low micromolar range. Upon further characterization, cyclisation was found to be essential for its activity. To increase metabolic stability, a series of disulfide mimetics were designed and a redox-stable 1,4-disubstituted 1,2,3-triazole analogue displayed activity in the double-digit micromolar range. Further experiments revealed that this triazole peptidomimetic is also active against CsrA from Escherichia coli and RsmA from Pseudomonas aeruginosa. This study provides an ideal starting point for medicinal chemistry optimization of this macrocyclic peptide and might pave the way towards broad-acting virulence modulators. [Display omitted] • Screening of self-designed phage library yielded very short macrocyclic peptide hits. • Identification of single-digit micromolar CsrA-RNA interaction inhibitor. • Synthesis of Triazole peptide as redox stable disulfide mimic. • NMR derived solution structure + docking experiments. • Activity on CsrA/RsmA from several species holds promise for broader acting antivirulence agents against Gram-negatives. [ABSTRACT FROM AUTHOR]

Summary Disulfide-rich peptides (DRPs) play diverse physiological roles and have emerged as attractive sources of pharmacological tools and drug leads. Here we describe the 3D structure of a centipede venom peptide, U-SLPTX 15 -Sm2a, whose family defines a unique class of one of the most widespread DRP folds known, the cystine-stabilized α/β fold (CSαβ). This class, which we have named the two-disulfide CSαβ fold (2ds-CSαβ), contains only two internal disulfide bonds as opposed to at least three in all other confirmed CSαβ peptides, and constitutes one of the major neurotoxic peptide families in centipede venoms. We show the 2ds-CSαβ is widely distributed outside centipedes and is likely an ancient fold predating the split between prokaryotes and eukaryotes. Our results provide insights into the ancient evolutionary history of a widespread DRP fold and highlight the usefulness of 3D structures as evolutionary tools. Graphical Abstract Highlights • A centipede toxin structure defines a distinct type of the CSαβ defensin fold • This fold is characterized by two disulfides versus at least three in other CSαβ peptides • 2ds-CSαβ peptides are widespread in both eukaryotes and prokaryotes • The 2ds-CSαβ fold probably has an ancient pre-eukaryotic origin Disulfide-rich peptides (DRPs) play many important physiological roles, and can be extremely taxonomically widespread. Here, Dash et al. show that a diverse centipede toxin family belongs to one of the most widespread DRP folds known, the cysteine-stabilized α/β fold, but that it represents a unique, ancient form of this fold. [ABSTRACT FROM AUTHOR]

Review: 128 refs. [ABSTRACT FROM AUTHOR]